Quantichrom nitric oxide assay kit

Manufactured by BioAssay Systems

Sourced in United States

The QuantiChrom Nitric Oxide Assay Kit is a colorimetric assay that quantifies nitric oxide (NO) levels in biological samples. It provides a simple, straightforward method to measure total nitric oxide, which includes both nitrite and nitrate.

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Lab products found in correlation

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Close spelling variants of this product

QuantiChrom kits (2 citations)

26 protocols using quantichrom nitric oxide assay kit

Measuring Intracellular Reactive Species in Fungi

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To measure the levels of the intracellular reactive species, C. albicans cells and A. niger spores in PBS (10⁸ mL⁻¹) were treated with Cu-MOFs (2 mg mL⁻¹) for 4 d as described in an earlier section. An untreated sample was used as a control. The treated and control cells and spores were washed with PBS once and incubated in PBS containing 10 mM of H₂DCFDA or DAF-FM DA at room temperature in the dark for 1 h. After incubation, the fungal cells and spores were washed with PBS twice and resuspended in 100 μL of PBS. The fungal suspensions were transferred to a black walled 96-well assay plate, and the fluorescence was measured at 495 nm (excitation) and 515 nm (emission) using a Synergy HTX Multi-Mode Reader (BioTek Instruments, VT, USA).
To measure the levels of H₂O₂ and NO_x in the media, 1 mL of PBS was treated with the Cu-MOFs (2, 1, 0.5, 0.25, 0.125, and 0 mg mL⁻¹) at room temperature with shaking. After treatment, the level of H₂O₂ and NO_x was measured immediately (0 d) and on the 4^th day. An Amplex™ Red Hydrogen Peroxide/Peroxidase Assay Kit (Molecular Probes, Eugene, OR, USA) and QuantiChrom™ Nitric Oxide Assay Kit (BioAssay Systems, Hayward, CA, USA) were used to determine the H₂O₂ and NO levels, respectively, following the manufacturer's protocols.

Veerana M., Kim H.C., Mitra S., Adhikari B.C., Park G., Huh S., Kim S.J, & Kim Y. (2021). Analysis of the effects of Cu-MOFs on fungal cell inactivation. RSC Advances, 11(2), 1057-1065.

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Cytokine-Induced Nitric Oxide Assay

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RAW 264.7 cells were stimulated with cytokines for 24 or 48 h. Nitrite concentration, a measure of NO production, was determined by Griess reaction using a QuantiChrom™ Nitric Oxide Assay kit (BioAssay Systems, Hayward, CA, USA). Briefly, 100 µl supernatant was mixed with an equal volume of Griess reagent (1% sulfanilamide, 5% phosphoric acid and 0.1% N-(1-naphthyl)-ethylenediamine), and the mixture was incubated at room temperature for 10 min. Absorbance was measured at a wavelength of 540 nm using a microplate reader (Molecular Devices Corporation, Sunnyvale, CA, USA). Nitrite concentration was then calculated from a NaNO₂ standard curve.

GAO Q., LIU Y., WU Y., ZHAO Q., WANG L., GAO S., WEN W., ZHANG W., GUO N., ZHOU J, & YUAN Z. (2015). IL-17 intensifies IFN-γ-induced NOS2 upregulation in RAW 264.7 cells by further activating STAT1 and NF-κB. International Journal of Molecular Medicine, 37(2), 347-358.

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Quantifying Reactive Species in PAM

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To estimate the concentration of reactive species in the medium after NTP exposure, 3 mL per well of complete MEM medium was exposed to soft jet plasma for 200 s in 12-well plates. Then, 100 uL was taken for each PAM group, and samples were processed promptly for H₂O₂ and NO_x detection following the manufacturer protocol for the QuantiChrom™ Peroxide Assay Kit (DIOX-250, BioAssay systems, Hayward, CA, USA) and the QuantiChrom Nitric Oxide Assay Kit (D2NO-100; BioAssay systems, Hayward, CA, USA), respectively. Absorbance was measured using the Synergy HT (BioTek Instruments) plate reader. Untreated complete medium was used as the control for estimation. To detect the intracellular RONS, 2 × 10⁵ cells from each group were seeded on the 4th day of the plasma treatment cycle of designated week time points in 35 mm dishes in triplicates. After 24 h, cells from all PAM groups were harvested and incubated with H₂-DCF-DA (D-399; Invitrogen, Waltham, MA, USA) reagent for 30 min. After incubation, cells were washed with 1X cold PBS, acquired immediately using a BD FACS Verse flow cytometer, and analyzed by FACS suite software.

Bhartiya P., Kaushik N., Nguyen L.N., Bekeschus S., Masur K., Weltmann K.D., Kaushik N.K, & Choi E.H. (2022). Periodic Exposure of Plasma-Activated Medium Alters Fibroblast Cellular Homoeostasis. International Journal of Molecular Sciences, 23(6), 3120.

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Comprehensive Nitric Oxide Analysis in PG-NOW

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The total NO_X (NO, NO₂, and NO₃) concentration in PG-NOW was determined by using a commercial QuantiChrom NO Assay Kit (QuantiChrom Nitric Oxide Assay Kit, BioAssay Systems, Hayward, CA, USA) based on the enhanced Griess method. All measurements were performed following the instruction of the manufacturers. The ORP and conductivity of the samples were analyzed by an ORP30 and CON30 Tester (Clean Instruments, Shanghai, China), respectively. The pH value of the PG-NOW samples was obtained using a pH spear (Eutech Instruments, Paisley, United Kingdom).

Borkar S.B., Negi M., Kaushik N., Abdul Munnaf S., Nguyen L.N., Choi E.H, & Kaushik N.K. (2023). Plasma-Generated Nitric Oxide Water Mediates Environmentally Transmitted Pathogenic Bacterial Inactivation via Intracellular Nitrosative Stress. International Journal of Molecular Sciences, 24(3), 1901.

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In Vitro Evaluation of Antiviral Compounds

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For virus culture, HCoV-229E was purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA) while MRC-5 human lung fibroblast host cells were purchased from the Korean Cell Line Bank (KCLB, Seoul, Korea). The QuantiChrom Nitric Oxide Assay Kit and QuantiChrom Peroxide Assay Kit (BioAssay Systems, Hayward, CA, USA) were used to measuring total NO and H₂O₂ levels, respectively, and the alamarBlue Assay Kit (Invitrogen, Waltham, MA, USA) was used to measure cell cytotoxicity. All procedures were performed according to the manufacturers' instructions. Chloroquine diphosphate and ritonavir were purchased from Sigma-Aldrich (Yongin, Korea). Stocks of these drugs were prepared in dimethyl sulfoxide (DMSO) and stored at −20 °C until further use. An antibody specific to the HCoV S protein was obtained from Sino Biological (Chesterbrook, PA, USA). All cell culture plasticware was purchased from SPL Life Sciences (Pocheon, Korea). Nitrogen (99.99%) and oxygen (99.99%) gases were purchased from Dong-A Scientific (Seoul, Korea). Other analytical reagent-grade solvents and chemicals were purchased from Sigma-Aldrich (Merck, St. Louis, MO, USA). Distilled water with a resistivity of 18.2 MΩ.Cm, obtained from a Milli-Q® water purification system (Millipore, Bedford, MA, USA) was used to prepare all aqueous solutions in this study.

Kaushik N.K., Bhartiya P., Kaushik N., Shin Y., Nguyen L.N., Park J.S., Kim D, & Choi E.H. (2022). Nitric-oxide enriched plasma-activated water inactivates 229E coronavirus and alters antiviral response genes in human lung host cells. Bioactive Materials, 19, 569-580.

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Antioxidant Enzyme Assays in Blood

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Following collection, the sample of whole blood was immediately stored at −20 °C for the measurement of GPx. For SOD measurement, red blood cells (RBC) were washed out of the sample of whole blood and stored at −20 °C. The remainder of the whole blood sample was centrifuged at 3000 rpm for 10 min at 4 °C and the resultant plasma was stored at −80 °C.
Whole blood GPx activity, RBC lysate SOD activity, plasma GR activity and plasma TAC concentration were measured using commercial assay kits (Randox Laboratories Ltd., Crumlin, UK) on an automated analyser (RX Daytona™; Randox Laboratories Ltd., Crumlin, UK). Plasma concentrations of MDA and HNE adducts were measured using an OxiSelect™ TBARS Assay Kit and an HNE Adduct Competitive ELISA Kit (Cell Biolabs Inc., San Diego, CA, USA), respectively, following the manufacturers’ instructions. A SPARK™ multimode microplate reader (Tecan, Grödig, Austria) was used to read the plates. Plasma NO concentration was measured using a QuantiChrom™ Nitric Oxide Assay Kit (BioAssay Systems, Hayward, CA, USA) according to the manufacturer’s instructions, and the plates were read on a Sunrise™ absorbance microplate reader (Tecan).

Moisejevs G., Bormane E., Trumpika D., Baufale R., Busmane I., Voicehovska J., Grigane A., Suba O., Silova A., Skesters A., Lejnieks A., Gailite L, & Brigis G. (2021). Glutathione Reductase Is Associated with the Clinical Outcome of Septic Shock in the Patients Treated Using Continuous Veno-Venous Haemofiltration. Medicina, 57(7), 689.

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Nitric Oxide Quantification in Serum

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Serum samples were thawed at room temperature and centrifuged at 14,000 rpm for 10 min at 4 °C before processing of samples for deproteination required for nitric oxide estimation. Briefly, deproteination was carried out by mixing 150 µl of serum with 8 µl of ZnSO₄. Subsequently, 8 µl of NaOH was added, vortexed and centrifuged at 14,000 rpm for 10 min at 4 °C. 100 µl of clear supernatant was used for estimating the nitric oxide. Circulating levels of nitric oxide were quantified in duplicate by a colorimetric method with a linear detection range of 0.6–200 µM using a QuantiChrom™ Nitric Oxide Assay Kit (Bioassay Systems, Hayward, CA) according to manufacturer's instructions and optical densities measured using a microplate reader set to 540 nm wavelength. The arithmetic mean of the duplicate samples was considered for analysis.

Kumar A., Singh K.P., Bali P., Anwar S., Kaul A., Singh O.P., Gupta B.K., Kumari N., Noor Alam M., Raziuddin M., Sinha M.P., Sharma A.K, & Sohail M. (2017). iNOS polymorphism modulates iNOS/NO expression via impaired antioxidant and ROS content in P. vivax and P. falciparum infection. Redox Biology, 15, 192-206.

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Measurement of Nitric Oxide Levels

Cited 1 time

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Nitric oxide production (total concentrations of nitrate and nitrite) was measured using a QuantiChrom Nitric Oxide Assay Kit (Bioassay Systems, Hayward, CA, USA), according to the Griess method [24 (link),25 (link)]. A sodium nitrite standard curve was prepared (0–150 μM). Deproteination of the plasma was accomplished by mixing 150 μl of plasma with 8 μl ZnSO₄, followed by mixing with 8 μl NaOH, vortexing, and centrifuging at 12,000 × g at 4°C for 10 min. Subsequently, 100 μl of each supernatant and the standard were added to 200 μl of the working reagent and incubated at 37°C for 60 min. After centrifugation of the reacted samples at 6,200 × g at 4°C for 5 min, the supernatant was diluted 2-fold with distilled water. Optical density was measured at 550 nm using an Eppendorf BioPhotometer Plus (Eppendorf, Hamburg, Germany). The total nitrite concentrations (μM) were determined according to the sodium nitrite standard curve.

Kume S., Yamato M., Tamura Y., Jin G., Nakano M., Miyashige Y., Eguchi A., Ogata Y., Goda N., Iwai K., Yamano E., Watanabe Y., Soga T, & Kataoka Y. (2015). Potential Biomarkers of Fatigue Identified by Plasma Metabolome Analysis in Rats. PLoS ONE, 10(3), e0120106.

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Cell Proliferation and NO Quantitation

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Relative cell proliferation was assessed using the Cell Counting Kit-8 (Dojindo
Laboratories) as described previously [5 (link)].
For drug testing, relative cell numbers were counted after treatment for 48 h
either with sorafenib (5 μM), with PD407824 (1 μM), with
Ellipticine (1 μM) or with combinations thereof. The cell numbers were
calculated by comparison to the control cells treated with DMSO. NO quantitation
in the cell lysate from cells treated with arginine-depletion, PTIO treatment,
or other compound treatments for 24 h was performed using the QuantiChrom Nitric
Oxide Assay Kit (BioAssay Systems, Hayward, CA, USA) according to the
manufacturer's instructions.

Kishikawa T., Otsuka M., Tan P.S., Ohno M., Sun X., Yoshikawa T., Shibata C., Takata A., Kojima K., Takehana K., Ohishi M., Ota S., Noyama T., Kondo Y., Sato M., Soga T., Hoshida Y, & Koike K. (2015). Decreased miR122 in hepatocellular carcinoma leads to chemoresistance with increased arginine. Oncotarget, 6(10), 8339-8352.

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Serum Arginase and Nitric Oxide

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Serum arginase activity and NO were measured using the QuantiChrom™ Arginase Assay Kit (BioAssay Systems, #DARG-100) and the QuantiChrom Nitric Oxide Assay Kit (BioAssay Systems, #D2NO-100), respectively, according to the manufacturer’s instructions.

Setoyama D., Matsushima T., Hayakawa K., Nakao T., Kanba S., Kang D, & Kato T.A. (2022). Blood metabolic signatures of hikikomori, pathological social withdrawal. Dialogues in Clinical Neuroscience, 23(1), 14-28.

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